Thrombus associated diseases are vascular conditions that are developed due to the presence of a clot. Such diseases are a major cause of mortality, and therefore developing thrombus-specific diagnosis, treatment, and detection methodologies and reagents is of great clinical importance. Pulmonary embolism (PE), deep-vein thrombosis, stroke, and atherosclerosis are examples of thrombus associated diseases.
Deep-vein thrombosis is a condition in which blood clots form in the deep blood vessels of the legs and groin. These clots can block the flow of blood from the legs back to the heart. Sometimes, a piece of a clot is detached and carried by the bloodstream through the heart to a blood vessel, where it lodges and reduces, or blocks, the flow of blood to a vascular tissue. This is called an embolism. Such a clot lodging in a blood vessel in the lung is a pulmonary embolism, or PE. PE can cause shortness of breath, chest pain, or even death.
In the United States alone, there are an estimated 600,000 patients that suffer pulmonary embolism each year. In approximately 378,000 of these patients, PE goes undetected, and approximately 114,000 of these patients die later due to complications associated with the disease. This high mortality is partly due to the absence of clinical symptoms in many cases and to the significant limitations associated with currently available methods of investigation and detection.
There is a need, therefore, for sensitive and effective assays to detect the presence of thromboembolism at various stages of development, for ways to diagnose the presence or absence of early and late thrombi, and for non-invasive reagents that can specifically bind thrombi and which will be useful for detecting the presence or absence of the early or late thrombus in patients.
Thrombus Formation
Crosslinked fibrin forms the underlying backbone of both venous and arterial clots or thrombi (Harker et al., Am. J. Cardiology, 60:20B-28B (1987)). Thrombi are formed when the enzyme thrombin is activated, leading to cleavage of plasma fibrinogen to release fibrinopeptides and expose a fibrin polymerization site (Hermans et al., Semin. Thromb. Hemost., 8:11-24 (1982)).
The biology of fibrin and clot formation has been investigated by many researchers in recent years, and a detailed understanding of the cascade of events leading to clot formation has emerged. There are two major activation pathways for coagulation: the intrinsic pathway which requires Factors XII, IX and VIII and the extrinsic pathway which involves tissue factor and Factor VII. Both pathways converge at the point of activating Factor X, the enzyme responsible for converting prothrombin to thrombin.
The extrinsic pathway is initiated by tissue factor, a ubiquitous cellular lipoprotein which forms a calcium-dependent complex with Factor VII. Upon complex formation, Factor VII is activated to Factor VIIa, which converts Factor X to Factor Xa. Factor Xa converts prothrombin to thrombin in conjunction with Factor Va, calcium and phospholipid. Prothrombin conversion also occurs on endothelial surfaces and activated platelets, and requires the assembly of a complex between Factor Xa, Factor Va, and prothrombin. This conversion requires the presence of phospholipid and calcium ions.
The intrinsic or contact coagulation pathway is initiated by platelets. The cascade begins with the formation of a complex among Factor XII, high molecular weight kininogen, and prekallikrein. Upon complex formation, Factor XII is cleaved to Factor XIIa. After the stepwise activation of Factors XI, IX, VIII, X, and V, as in the extrinsic pathway, prothrombin is activated to thrombin. Thrombin, which is a trypsin-like serine protease, is the central regulator of hemostasis and thrombosis. Fibrin is derived from fibrinogen, and polymerization of fibrin occurs following enzymatic cleavage of fibrinogen by thrombin. Fibrinogen (340 kD) consists of three pairs of identical peptides, designated Aα, Bβ, and γ. Chemical structural analysis and electron microscopy have demonstrated that the protein has a trinodular structure. Two AαBβγ subunits are oriented in an antiparallel configuration. The amino terminal portions of the six chains are bundled together in a central “E” domain. Two coiled-coil strands extend outward from either side of the E domain to the two terminal nodes, the “D” domains. These coiled coil regions are 110 amino acids long and composed of all three chains. The D domains contain two high affinity Ca2+ binding sites and are involved with the E domain in fibrin polymerization. Extensive disulfide bridges covalently crosslink the two subunits, and stabilize the globular domains. The C-terminal portions of the Aα chains form flexible extensions beyond the D domains. The D domain contains Factor XIIIa crosslinking sites and is the primary site of plasmic digestion during fibrinolysis.
Fibrin formation from fibrinogen is a spontaneous self-assembly process resulting from the removal of fibrinopeptides by thrombin. Thrombin cleavage at the Arg16-Arg17 bond in the Aα chains and at the Arg14-Gly15 bond on the Bβ chains releases fibrinopeptides A and B, and exposes a polymerization site in the E domain consisting mainly of the N-terminus of the α chain. This N-terminus, which bears the sequence Gly-Pro-Arg-Val, binds to a complementary polymerization site on two adjacent fibrinogen chains. End to end association of these fibrinogen molecules mediated by the D domains, creates a binding site for the E domain polymerization site, located on a third fibrinogen molecule. This DD(E) ternary complex forms a core that stabilizes the forming fibrin gel. The initial polymerization product is a linear, two-stranded protofibril. Lateral coalescence of these protofibrils results in thick fibers and a branched, three dimensional matrix. Lateral assembly is complex but probably involves the B polymerization site (the N-terminus of β) and trimolecular complexes formed through D domain interactions.
Adjacent fibrin monomers within the fibrils become covalently crosslinked by Factor XIIIa, a plasma transglutaminase which is itself activated by thrombin and fibrin. These crosslinks add mechanical stability to the fibrin network and increase resistance to clot degradation. Factor XIIIa also enhances clot stability by crosslinking specialized proteins to fibrin, including the plasmin inhibitor α2 antiplasmin, and the adhesion protein fibronectin.
Thrombus Imaging
The search for thrombus-specific imaging agents began three decades ago when radiolabeled fibrinogen was first evaluated (Kakkar et al., Lancet, 1:540-542 (1970)). Since then a number of thrombus imaging agents have been described, including agents that are incorporated into forming thrombi and agents that bind to components of previously formed thrombi (Knight et al., Radiology, 156:509-514 (1985); Alavi et al., Radiology, 175:79-85 (1990); Rosebrough et al., J. Nuc. Med. 31:1048-1054 (1990)). Among the recent approaches that have been taken in the development of materials useful for visualizing or imaging thrombi are radiolabeled platelets and anti-platelet antibodies that bind to forming thrombi, anti-fibrin antibodies, anti-activated platelet antibodies, and activated or inactivated tissue type plasminogen activator (tPA) (Thakur et al., Throm. Res., 9:345-357 (1976); Palabrica et al., Proc. Natl. Acad. Sci., 86:1036-1040 (1989)).
Platelet affinity peptides have also been used to detect clots. This approach utilizes small 99mTc-labeled peptides capable of binding to platelets. The platelets, with labeled peptide attached, become incorporated into thrombi and render the thrombi detectable (Bautovich et al., J. Nucl. Med., 35:195-202 (1994); Muto et al., Radiology, 189 (suppl):303(1993)).
Because platelets in thrombi degrade over time, the use of platelet affinity peptides, anti-platelet antibodies and other agents that bind to platelets or that detect platelet location are only useful for detection of early clots (less than 12 hours) and cannot be used in detection and imaging of embolism, particularly pulmonary embolism.
Since Fibrin is the major protein component in thrombi it is thus a desirable target for agents that can mark the location and gauge the size of a clot in a subject. Fibrin targeting, however, is complicated by the close structural similarity between fibrin and its circulating precursor, fibrinogen. One successful approach has involved the isolation of monoclonal antibodies specific to fibrin. One such class of monoclonals recognizes the newly exposed N-termini of the α and β chains of the fibrin monomers. Another class of monoclonal antibodies recognizes epitopes exposed as a result of polymerization, such as the covalent crosslinks formed by Factor XIII, the DD dimer domain, or the putative tPA binding site. The use of antibodies as imaging agents does, however, have some disadvantages: The high molecular weight of antibodies necessitates that a larger mass of agent must be delivered to a clot than would be required of a small molecule, and this may be a serious limitation when higher concentrations of an imaging agent are essential to obtaining adequate signal contrast. Labeled antibodies often present clearance problems because of relatively long circulating half-lives in vivo, limiting contrast with the blood and tissue background. In addition, antibodies are often expensive to prepare and formulate, and their use can lead to undesirable and potentially fatal immunogenic responses.
Another method used for pulmonary embolism diagnosis is the ventilation/perfusion scan. In a ventilation/perfusion scan, the patient inhales a radiographic gas, and images of regions of the lung that are capable of ventilation are recorded. Subsequently, the patient is injected with a radioactive agent and the movement of the agent through the pulmonary artery is traced. The two images are compared, and any area of thrombosis is detected by contrasting the ventilation data with the perfusion data. Approximately 930,000 ventilation/perfusion scans are performed every year in the United States, but approximately 60% are inconclusive.
An alternate method for pulmonary embolism diagnosis is x-ray angiography. This method is performed by introducing an x-ray opaque (radiopaque) compound proximally to the heart or pulmonary artery via arterial catheter introduced through the patient's femoral vein. The compound is traced through the pulmonary artery by an x-ray camera and thrombosis is detected by such tracing. Although this method is considered a “gold standard” test by clinicians and approximately 60,000 angiographies are performed annually in the United States, the test is invasive and expensive. Moreover, 1 out of 200 patients undergoing the x-ray angiography dies as a direct result of the procedure itself.
Recently, fibrin binding polypeptides have been discovered that are useful, when detectably labeled (e.g., with a paramagnetic metal or radionuclide), as imaging agents for localization and imaging of fibrin clots. See, PCT/US00/20612, incorporated herein by reference. Such fibrin binding polypeptides represent a much needed advance in the art, however there is additional room for improvement in features such as the avidity of the polypeptides for fibrin substrates.
A group of polypeptides has now been discovered which bind to fibrin and also exhibit a low “off-rate”, that is, they have a lower dissociation rate than previously characterized binding moieties for fibrin. Such slow-dissociating fibrin binding polypeptides will concentrate at the sites of fibrin clots and remain there longer in comparison to freely circulating such polypeptides, which means that after administration to a patient the circulating polypeptides unbound to fibrin will be cleared from circulation and the remaining polypeptide in a patient's system will be primarily polypeptide that is bound to fibrin. Thus, interfering background signal attributable to circulating labeled polypeptide is cleared and labeled polypeptide at the site of a clot remains, creating a better detectable signal for localization and imaging of a clot.
The newly discovered fibrin binding polypeptides have amino acid sequences differing from previously described fibrin binding moieties. The preparation and use of such polypeptides, for example as imaging agents or as fusion partners for fibrin-homing therapeutics, is described in detail herein.